JP2017072586A - Locally imaging structure in sample at high spatial resolution - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable high spatial resolution imaging of a structure in a sample.SOLUTION: A structure is marked with luminescence markers, and light having an effect on emission of luminescence light by the luminescence markers is directed onto a sample with an intensity distribution having a zero point and intensity maxima neighboring the zero point in at least one direction. A scan area, which is a part of the sample, is scanned with the zero point. Luminescence light emitted out of a local area including the zero point is registered and assigned to a respective location of the zero point in the sample. Dimensions of the scan area in at least one direction, in which the intensity maxima are neighboring the zero point, are limited such that they are not larger than 75% of a distance of the intensity maxima in the at least one direction.SELECTED DRAWING: None

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is based on German Patent Application No. 10 2015 116 023.4 filed on September 22, 2015 and German Patent Application No. 10 2016 filed on March 7, 2016. 104 claims the priority of 100.9.

  The present disclosure relates to a method for spatial high resolution imaging of structures in a sample where the structures are marked with luminescence markers. The present disclosure further relates to a scanning luminescence optical microscope for performing this method.

  The present invention belongs to the field of high-resolution scanning luminescence optical microscopy, where the wavelength of luminescence light emitted from each sample and the emission of luminescence light in a spatially limited area. Measures are taken that allow luminescent light to be assigned to locations in the sample with higher spatial resolution than the diffraction barrier at the wavelength of any excitation light by which the luminescent marker is excited. Often, a luminescent marker is a fluorescent marker that emits fluorescent light as luminescent light after excitation by excitation light. In that case, one refers to fluorescence microscopy.

  Known methods for spatial high-resolution imaging of structures in samples, where structures are marked with luminescence markers, and scanning luminescence optical microscopy, approach zeros and zeros to increase spatial resolution With the intensity distribution having a maximum intensity value, light that influences the emission of luminescent light is directed onto the sample by the luminescence marker. Often this light is luminescence-suppressing light that suppresses the emission of luminescent light by all those luminescent markers outside the zero. The luminescent light emitted from the sample can therefore be assigned to the location of the zero point in the sample, since only the luminescent markers located there can emit luminescent light.

  In STED fluorescence microscopy, for example, a fluorescent marker previously excited by excitation light is such that only the fluorescent marker in the zero point area may emit fluorescence light measured thereafter. Except for the fluorescent marker in the area, reverse excitation is again performed by the induced light as the fluorescence suppression light. This fluorescent light can therefore be assigned to the location of the zero point in the sample. The spatial distribution of fluorescent markers within the sample is determined by scanning the sample at zero. In this way, the shape and spatial distribution of the structures in the sample that are marked with fluorescent markers can be imaged.

  In GSD fluorescence microscopy, fluorescence suppression light shifts the fluorescent markers to an electronic dark state so that the fluorescent markers outside the zero area are no longer excitable due to the emission of fluorescent light by the excitation light.

  In RESOLFT fluorescence microscopy, fluorescence suppression light that moves the photochromic fluorescent marker from the fluorescent state to the non-fluorescent state is used except for the fluorescent marker in the zero point area. When the fluorescent marker subsequently receives excitation light, only the fluorescent marker in the area of the zero point of the intensity distribution of the fluorescence suppression light is excited for emission of the fluorescent light by the excitation light. Thus, the fluorescent light emitted by the fluorescent marker in the sample can also be assigned here to the location of the zero point of the intensity distribution of the fluorescence suppression light in the sample.

  In all the methods of high spatial resolution scanning luminescence optical microscopy described so far, the luminescent markers in the respective samples can be used so that the luminescent markers can no longer emit luminescent light. There is an inherent danger of bleaching temporarily or even permanently, ie deactivating their luminescent markers. This danger is due to stopping all luminescent markers outside the zero point area from emission of luminescent light, and strongly increasing the size of the zero point area from which the luminescent marker can still emit luminescent light. This is due to the fact that the intensity of the luminescence suppression light needs to be extremely high. At this high intensity, when the zero point area of the luminescence suppression light comes closer to the luminescent marker, i.e. before the luminescent marker enters the zero point area for the first time and therefore by the first registered luminescence marker, Already before the emission of luminescence, the luminescence suppression light has already stressed the luminescence markers in the sample. This is because luminescent markers that have a tendency to bleach are desirable to maximize spatial resolution, so they may not be used at all in the described method, or at least high in luminescence-suppressing light. It can have the result that it may not be used with strength.

  Several approaches have been pursued to avoid the described problems of temporary bleaching and particularly permanent bleaching in high resolution scanning luminescence optical microscopy. DE 10 2005 027 896 and U.S. Pat. No. 7,719,679 belonging to the same patent family show that the same area of the sample is close to the zero point with an optimized time repetition interval. STED fluorescence microscopy in pulse while scanning each sample at a relatively long time interval or at the zero point of the intensity distribution of the guide light so as to receive only the high intensity of fluorescence suppression light at the maximum value Teaches the application of guided light to the sample. In this way, the fluorescence intensity light obtainable from the sample is increased since the rate at which the fluorescent marker enters the dark state with only permanent decay or only slow decay from further excitation by the induced light is significantly reduced. In other words, the relatively long repetition interval at which each individual area of the sample receives the intensity distribution of the fluorescence suppression light maximizes the overall amount of fluorescent light that can be obtained from the entire sample within a period of time. This procedure also reduces the tendency of the fluorescent marker to bleach because a higher distribution of excited states from which photochemical destruction of the fluorescent marker can occur is avoided.

  In order to perform high spatial resolution fluorescence microscopy even with fluorescent markers that tend to bleach, DE 10 2011 051 086 and US 2014/0097358, which belong to the same patent family, In addition to the scanning speed at which the sample is scanned and the light intensity of the fluorescence suppression light intensity distribution, the fluorescence light is emitted from the zero area of the fluorescence suppression light intensity distribution as individually detectable photons. Teaches adjusting the scanning conditions relative to each other, including the properties and concentration of the fluorescent markers in the sample. The image of the structure in the sample, marked with a fluorescent marker, is then composed of zero locations, which are assigned detected photons during several iterations of scanning the sample at zero. . In this way, the probability of bleaching the fluorescent marker is reduced before the fluorescent marker reaches zero and is therefore measured for the first time. This is due to the fact that the probability of bleaching correlates with the intensity of fluorescent light obtained from individual fluorescent markers. Since fluorescent light is minimized to individual photons, the risk of bleaching is also minimized. In general, however, in the known methods from DE 10 2011 051 086 and U.S. Patent Application 2014/0097358, the zero of the fluorescence suppression light is a fluorescence suppression close to the zero. It still only reaches the individual fluorescent markers after the zero has previously received a high intensity in the area of maximum light intensity.

  Since it is also possible to use materials that are susceptible to bleaching in high spatial resolution scanning luminescence optical microscopy, International Patent Application Publication No. 2011/131159 and US Pat. It is known from US Pat. No. 024,279 that the structure of interest moves the measurement front over a sample marked with a luminescence marker. At the measurement front, the intensity of the light signal is reduced by a portion of the luminescent marker that emits luminescent light by first moving the luminescent marker to the luminescent state and then moving the luminescent marker to the non-luminescent state. In such a way that it is increased starting from presence and then reduced back to non-existence, it increases over the depth of the measurement front, which is smaller than the diffraction barrier, at the wavelength of the optical signal. Luminescent light from the area of the measurement front is registered and assigned to each position of the measurement front in the sample. The assignment of luminescent light to a location along the measurement front can also be achieved by assigning registered photons to a single luminescent marker, for example in the same way as in optical microscopy known as GSDIM. Can be performed at spatial resolutions exceeding.

  An option to increase the speed of imaging the structure of interest in a sample in scanning luminescence optical microscopy is to scan the sample in parallel with multiple zeros of luminescence suppression light. Here, the luminescence light emitted from the sample is assigned separately to each zero point of the luminescence suppression light. From German Patent No. 10 2006 009 833 and US Pat. Nos. 7,903,247 and 7,646,481 belonging to the same patent family, two orthogonal line patterns of luminescence suppression light It is known that the intensity distribution of the luminescence suppression light is formed by a grid of zero points in that it is superimposed on the sample. Interference between the light of the two line patterns is avoided so that the intensity distributions of those patterns are simply added. The desired zero of the intensity distribution of the luminescence suppression light remains at the intersection of the line shaped zeros of both line gratings, which are defined by the adjacent intensity maximum of the luminescence suppression light. To completely scan a sample in an area of a grid of zeros, it is sufficient to shift each zero over the distance to its nearest neighbor in the two directions of the two line patterns. Again, the majority of the luminescence markers in the sample are subjected to a high intensity of luminescence suppression light before one of the zeros reaches the luminescence marker so that the luminescence marker is registered for the first time. Therefore, luminescent markers should be selected so that they can withstand these high light intensities without bleaching.

  Li D et al .: Extended-resolution structured illumination imaging of endocetic and citoskeletal dynamics, Science 2015 Aug 28; 349 (6251) The sample is marked with a fluorescent marker, and the sample is continuously scanned with a line or plane zero that matches the light intensity distribution of the fluorescence activation light and fluorescence suppression light in different directions, and the fluorescent light emitted by the sample is a camera It is registered with. By evaluating the registered light intensity, an image of the structure of interest in the sample can be reconstructed, and the spatial resolution of that image matches the fluorescence activation light and the fluorescence excitation light, where no fluorescent light is emitted from the sample. It is increased due to narrowing the zero point. Further, in this known method, the zero point of the fluorescence activation light and the fluorescence excitation light that simultaneously serves as the fluorescence deactivation light is defined by the intensity maximum values of the fluorescence activation light and the fluorescence excitation light. All luminescent markers in the sample will have their fluorescence activation light and fluorescence in these intensity maximum areas before they enter the zero area where the fluorescence activation light and fluorescence excitation light coincide. Receives high intensity of excitation light. Therefore, the risk of bleaching the fluorescent marker before it contributes to the relevant measurement signal is also very high with this known method.

  WO 2014/108455 and US Pat. No. 9,267,888, which belong to the same patent family, disclose a method for high spatial resolution imaging of structures in a sample, wherein the structures are luminescent. Marked with a sense marker, the sample receives excitation light and receives induced light as luminescence suppression light, as in STED fluorescence microscopy, and the area of the sample is reduced and emitted from the sample to that area and detected The fluorescent light can be assigned to the area of the zero point of the guide light. In order to protect the luminescent marker against the high intensity of the guided light in the area of its maximum value close to the zero, the sample is an excitation-suppressed light whose intensity distribution has a local minimum corresponding to the zero of the guided light Receive more. This excitation suppression light may in particular be a switch-off light that switches a switchable luminescence marker that is outside the minimum value of the excitation suppression light to an inactive state, in which these luminescence markers are excited Not excitable due to the emission of fluorescent light by light. In particular, the luminescent markers can be switchable fluorescent dyes such that they are used in high spatial resolution RESOLFT fluorescence microscopy. In known methods from WO 2014/108455 and U.S. Pat. No. 9,267,888, however, the switchability of luminescent markers is to increase spatial resolution. Rather, it is mainly used to protect luminescent markers against bleaching due to high intensity of induced light.

  RA Hoeve et al .: Controlled light-exposure microscopic reduces photobleaching and phototoxicity in live-cell imaging, Nature Biotechnol 25, Nature Biotechnol. 2, February 2007, pages 249-253 disclose a method of confocal fluorescence microscopy, where the sample is scanned with focused excitation light to image the structure in the sample, and the structure is a luminescent marker. Marked with Here, as soon as the number of photons emitted by the excitation luminescence marker in the sample and registered by the detector reaches the upper threshold corresponding to the desired signal-to-noise ratio, the excitation light will be reflected in the sample. It is switched off at each position of the focused excitation light. If the number of emitted and registered photons does not reach the lower threshold within a predetermined portion of the maximum pixel dwell time, this is related to the luminescent marker in the sample at each position of the focused excitation light. The excitation light is still switched off because it indicates that no concentration is found. In this way, the loading of the sample by the excitation light is significantly reduced compared to applying the same amount of light to the sample at each location.

  T. T. Staudt et al .: Far-field optical nanoscopic with reduced number of state transition cycles, Optics Express Vol. 19, no. 6, March 14, 2011, pages 5644-5657, discloses a method called RESCue-STED, which transfers the method described by RA Hoeve et al. For confocal fluorescence microscopy to STED fluorescence microscopy. Here, the sample only receives a high intensity of guided light as long as necessary or preferred.

German Patent Application No. 10 2005 027 896 US Pat. No. 7,719,679 German Patent Application Publication No. 10 2011 051 086 US Patent Application Publication No. 2014/0097358 International Patent Application Publication No. 2011-131590 US Pat. No. 9,024,279 German Patent No. 10 2006 009 833 US Pat. No. 7,903,247 US Pat. No. 7,646,481 International Patent Application Publication No. 2014/108455 Specification US Pat. No. 9,267,888

Li D, et al .: Extended-resolution structured imaging imaging of endocetic and scientific dynamics, Science 2015 Aug 28; 349 (6251) RA Hoeve et al .: Controlled light-exposure microscopic reduces photobleaching and phototoxicity in live-cell imaging, Nature Biotechnol 25, Nature Biotechnol. 2, February 2007, pages 249-253 T. T. Staudt et al .: Far-field optical nanoscopic with reduced number of state transition cycles, Optics Express Vol. 19, no. 6, March 14, 2011, pages 5644-5657

  There remains a need for a method of high spatial resolution imaging of structures in a sample where the structures are marked with luminescence markers, and a scanning luminescence optical microscope to perform such methods, for example, biology High light intensity so that even luminescent markers sensitive to high light intensity can be used and / or structures in each sample can be repeatedly imaged to monitor structural changes during the process. The load on the luminescent marker in the sample due to intensity is generally reduced.

  The present invention provides a method for high resolution imaging of structures in a sample in which the structures are marked with luminescent markers. The method includes directing light on a sample that influences the emission of luminescent light with a luminescence marker in an intensity distribution having a zero and an intensity maximum close to the zero in at least one direction; scanning at the zero Scanning the area, where the scanning area is part of the sample; while scanning the scanning area, register the luminescent light emitted from the local area including the zero in the sample And assigning the registered luminescent light to each location of the zero in the sample. The dimensions of the scanning area in at least one direction where the intensity maximum is close to the zero point in the sample are limited such that those dimensions do not exceed 75% of the distance of the intensity maximum in at least one direction.

  The present invention also provides a further method for high spatial resolution imaging of structures in a sample where the structures are marked with luminescent markers. The method includes directing light on a sample that influences the emission of luminescent light by means of a luminescent marker in an intensity distribution having a zero and an intensity maximum close to the zero in at least one direction; scanning at the zero Scanning the area, where the scanning area is part of the sample; while scanning the scanning area, register the luminescent light emitted from the local area including the zero in the sample And assigning the registered luminescent light to each location of the zero in the sample. Here, the scanning area is scanned at the zero point starting at the center point and with increasing distance to the center point in at least one direction.

  The present invention further includes a light source configured to provide light; a light shaper configured to direct light onto the sample with an intensity distribution having a zero and an intensity maximum proximate to the zero; and the zero A scanner configured to scan a scanning area, wherein the scanning area is a partial area of the sample; and a detector configured to register luminescent light emitted from the zero point area; A scanning luminescence optical microscope comprising a controller programmed with software implementing at least one of the above methods.

  Other features and advantages of the present invention will become apparent to those skilled in the art upon consideration of the following drawings and detailed description. All such additional features and advantages are intended to be included herein within the scope of the present invention as defined by the claims.

  The invention can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present invention. In the drawings, like reference numerals designate corresponding parts throughout the several views.

Intensity distribution of excitation light and fluorescence suppression light as an example of light that affects the emission of luminescent light by the luminescent marker in the sample, and the resulting effective excitation of the fluorescent marker in the sample for emission of fluorescent light Is shown schematically. 1 shows the dimensions of the scanning area of a sample to be scanned with the intensity distribution according to FIG. 1 in the method according to the invention for the intensity distribution of fluorescence suppression light according to FIG. FIG. 6 is a schematic view of a scanning area of a sample to be scanned in a top view, with a scan along a meander-shaped course. FIG. 2 is a schematic view of a scanning area of a sample to be scanned in a top view, where scanning is performed along a spiral course. FIG. 6 is a schematic view of a scanning area of a sample to be scanned in a top view in another method according to the present invention, where scanning is also performed along a spiral course. 1 schematically shows a fluorescence microscope as an example of a scanning luminescence optical microscope according to the present invention. FIG. 7A shows a confocal image of the sample taken on the upfront, and FIG. 7B shows a confocal image of the sample taken on the upfront. Fig. 4 shows a partial image of a sample taken according to the invention after an individual scanning area of the sample has been selected based on a confocal image. Fig. 4 shows the dependence between the number of images that can be taken successively in the scanning area of the sample and the dimensions of the scanning area. FIG. 7 schematically shows another fluorescence microscope not shown in FIG. 6 as a further exemplary embodiment of a scanning luminescence microscope according to the invention. FIG. 10 is a schematic view of a scanning area of a sample to be scanned with an additional view of an adjacent area where switch-off light is directed onto the sample by the scanning fluorescence microscope of FIG. FIG. 11 shows the arrangement of several scan areas in a sample that should be scanned according to FIG. 10 in order to scan the sample in the scan area. Fig. 4 shows another arrangement of scanning areas in a sample. 2 is a block diagram of an embodiment of a method according to the invention; FIG. 6 is a block diagram of another embodiment of a method according to the present invention.

  In the method according to the invention for spatially high-resolution imaging of a structure in a sample, the structure is marked with a luminescence marker, with an intensity distribution having a zero and an intensity maximum close to the zero, the emission of fluorescent light by the luminescence marker. Light affecting the sample is directed onto the sample. The scan area, which is a partial area of the sample, is scanned at the zero point, and the luminescent light emitted from the local area including the zero point is registered, and when the luminescent light is registered, the zero point was in the sample, respectively. Assigned to a location. The size of the scanning area is limited to at most 75% of the distance of the intensity maximum in each direction, in at least one direction where the intensity maximum is close to the zero and in particular in any direction.

  Insofar as luminescent markers are mentioned herein, the luminescent markers may in particular be fluorescent markers. However, other luminescence markers whose luminescence properties can be based, for example, on chemiluminescence or electroluminescence can also be used. This includes that the excitation of luminescent markers for emitted luminescent light is not limited to a particular mechanism in the method according to the invention. However, often the excitation of the luminescent marker for the emission of luminescent light is obtained by excitation light.

  The light that affects the emission of luminescent light by the luminescent marker is such that the luminescence suppression light reverses the luminescent marker that has been excited, for example, by moving the luminescent marker to the dark state or by stimulated emission. In this respect, it can be luminescence suppression light that suppresses the emission of luminescence light by the luminescence marker in that the luminescence marker suppresses the emission of luminescence light. Further, the light that affects the emission of luminescent light by the luminescent marker can be light that transfers the luminescent marker from one non-luminescent state to a further non-luminescent state that is particularly well protected against bleaching. . Furthermore, the light that affects the emission of luminescent light by the luminescent marker is such that when that light is combined with other light, this emission occurs only from the zero area of the light intensity distribution. The light can only affect the emission of luminescent light.

  In another embodiment, the light that affects the emission of luminescent light by the luminescent marker is such that the luminescent capable light excites the luminescent marker for luminescence or causes the luminescent marker to go from a dark state, for example. It is a luminescent capable light that allows the emission of luminescent light by means of a luminescent marker in that it moves to an excitable state.

  The zero point of the light intensity distribution that affects the emission of luminescent light by the luminescence marker is at least the local intensity minimum value of the light intensity distribution. Often, the zero is the intensity minimum at which the light intensity drops to essentially zero. In the ideal case, the light intensity actually drops to zero at the center of the zero. However, this is not an essential requirement. For example, if the light is luminescence-suppressed light, the intensity of the luminescence-suppressed light is very low in the zero-point area, and therefore there is no or at least essentially no luminescence suppression of the luminescence marker, That is, it is sufficient if there is at least no dominant suppression. The zero is then defined by the area where the luminescence suppression light at least essentially suppresses the emission of luminescence light by the luminescence marker. Everything between these areas in which the luminescence suppression light at least essentially suppresses the emission of luminescence light by the luminescence marker is referred to herein as a “zero point” or “zero point area”.

  When an intensity maximum close to the zero point of the light intensity distribution that affects the emission of luminescent light by a luminescent marker is referred to in the plural form herein, this means that the zero point is a ring around the zero point. It does not exclude the case surrounded by the expanding maximum intensity value. In each virtual section through the light intensity distribution that affects the emission of luminescent light, such a ring-shaped intensity maximum appears in the form of two intensity maximums close to the zero on either side of the section.

  The intensity maximum may be close to the zero in one, two or three directions. Thus, the zero can be a planar shape, a line shape, or a point shape. A zero can intersect a two-dimensional or one-dimensional sample in such a way that even a line-shaped or point-shaped zero is defined by an intensity maximum in all directions of the sample's main extension. Furthermore, the scanning area is not defined by intensity maxima in all directions of the main extension of the sample with different orientations of the zeros in order to maximize the spatial resolution of the imaging in all directions of the main extension of the sample. It can be scanned continuously at zero. The physical dimensions of the scanning area are limited in at least one direction, preferably in all directions, where the intensity maximum is close to the zero in the sample.

  The intensity maxima close to the zeros in the sample is often much higher than the area of the light intensity distribution that affects the emission of luminescent light by the luminescence marker, and these areas are The light already has the desired effect on this emission in the area by the luminescence marker, in that the light suppresses the emission of the luminescent light, for example. The very high intensity of the intensity maximum is a result of the overall high intensity of the light, which on the other hand is the zero point in which the light has at least essentially no influence on the emission of the luminescent light by the luminescence marker. It is a prerequisite for the area to be strongly and spatially defined. As a result, there is an intermediate area between the area of the light intensity distribution that directly defines the zero point area and the area of the intensity maximum value of the intensity distribution, in which the light intensity is within the intensity maximum value. It remains far below the intensity. These intermediate areas are intentionally used in the method according to the invention in that the dimensions of the scanning area do not exceed 75% of the distance of the intensity maximum in each direction. If the dimensions of the scan area remain less than 50% of the maximum intensity distance in each direction, the points in the scan area do not receive the full intensity of the light intensity maximum. However, even at the 75% limit of distance, there is a significant limitation on the average intensity of light that affects the emission of luminescent light received by the scanning area. It should be understood that the average intensity of light received by the scanning area decreases as the size of the scanning area remains smaller than the distance of maximum intensity in each direction. From an absolute point of view, the dimension of the scanning area in each direction between the intensity maxima can be up to 300 nm. Preferably, the dimension in each direction is at most 200 nm, more preferably the dimension is about 100 nm. These absolute numbers relate to the wavelength of the luminescent light, the wavelength of the light that affects the emission of the luminescent light by the luminescent marker, and / or the wavelength of the possible excitation light, all in the visible range.

  The scanning area is only the partial area of the sample that is scanned when carrying out the method according to the invention, correspondingly to the area of interest in the sample, i.e. the attention of structures marked with luminescence markers in the sample. It can be a partial area of the sample, which should be led to the details to be done.

  The present invention intentionally allows the partial area of the sample that is scanned and thus imaged to remain small. Often, the scanning area extends over a distance of the size of the diffraction barrier at the wavelength of light that affects the emission of luminescent light by the luminescent marker. On the other hand, due to the substantial reduction of the average light intensity experienced by the scanning area, even luminescent markers that have a strong tendency to bleach can be used successfully, or the scanning area in the sample can be repeatedly scanned at the zero point of the light intensity distribution It is.

  The option of repeatedly scanning the scan area in the sample at a high repetition rate at the zero point of the intensity distribution also allows a dynamic process in the structure of interest in the sample to be solved in time. The luminescent marker in the sample has a particularly low tendency to bleach so that a specific high number of photons are obtained from each individual fluorescent marker in the scanning area by the method according to the invention, so that especially the scanning of the sample Many images of the area can be taken and thus even long-term variations of the structures of interest in the sample can be observed. Generally, the scan area is scanned at 100 × 100 = 10,000 or less image points. This is possible within a few milliseconds. Therefore, an image frequency of 100 Hz or higher can be realized.

  Advantageously, in each direction where the intensity maximum is close to the zero in the sample, the size of the scan area is not greater than 45%, 25% or even 10% of the distance of the intensity maximum in each direction. Regarding the intensity of light that affects the emission of luminescent light by the luminescence marker, the size of the partial area of the sample in each direction where the intensity maximum is close to the zero in the sample is the intensity of the light starting at the zero. It is advantageous if it is not greater than the distance increasing over it in each direction up to 50%, 25%, 10% or 5% of the light intensity at the closest intensity maximum. Correspondingly, the maximum load of the luminescent marker in the scanning area is limited to 50%, 25%, 10% or 5% of the light intensity at the adjacent intensity maximum.

  The same observation on which the embodiments of the invention described so far are based is also different from the method according to the invention in that the scanning area of the sample is scanned at the center point and with increasing distance to this center point. Used in the embodiment. As long as the distance to the center point remains less than 75% of the distance of the maximum intensity close to the zero point in the sample in the same direction, by the light that affects the emission of fluorescent light by the fluorescent marker on the scanning area The average load remains small compared to known scanning luminescence optical microscopy methods. In either case, the luminescent marker near the center point is registered with respect to the location of the luminescent marker in the sample prior to receiving a higher intensity of light that affects the emission of luminescent light. Thus, for luminescent markers that tend to bleach, the relative intensity of the registered luminescent light, especially if the distance to the center point increases to and beyond the maximum intensity distance in each direction, It can decrease with increasing this distance to the center point. Nevertheless, a partial area of the sample directly around the center point is scanned and imaged with a high yield of luminescent light.

  The last described embodiment of the method according to the invention can in fact be realized in that the scanning area is scanned along a spiral course around the center point.

  In all embodiments of the method according to the invention, prior to scanning the partial area of the sample to be scanned, it is marked with a luminescent marker in another way to determine the partial area of the sample to be scanned It is often preferred to image the structure. In general, the sample to be scanned or a partial area of the scanning area is the focus of the sample in which specific details of the structure exist or specific development of the structure occurs in a biological process. It is a partial area. This primary imaging can be performed under local or large area excitation of luminescent markers for emission of fluorescent light and without using light that affects the emission of luminescent light.

  Prior to scanning the scanning area, the larger area of the sample has been reduced by at least 50% of the light that affects the emission of luminescent light to determine the position of the partial area to be scanned in the sample. It can be scanned with intensity and / or with a scanning speed increased by at least 50%. In this primary scan, all points in the larger area of the sample receive a high intensity of light in the area of maximum intensity. However, this intensity is deliberately reduced and / or only affects the luminescent marker over a shorter period of time.

  In one embodiment, another scanner is used to scan a larger area of the sample than when scanning the scan area.

  When scanning different areas to scan a larger area of the sample to determine the scanning area, and subsequently to scan the scanning area, especially for scanning a very limited or small scanning area. A suitable scanner can be used. Due to the small size of the scanning area, these scanners do not allow greater movement of the light intensity distribution with zeros relative to the sample, but can be scanners that achieve possible movement very fast and / or accurately. Thus, the scanning area can be scanned at a high repetition rate, for example, to monitor fast changes in the structure of interest in the sample in the scanning area.

  In particular, the sample stage or sample holder for the sample can be moved in at least one direction relative to the objective lens by which light is directed onto the sample, but in order to scan the scanning area in at least one direction, Electro-optic scanners, acousto-optic deflectors or galvo scanners or galvo mirrors, ie deflecting mirrors with galvometric drives are used. The scanner for the scanning area can be combined with an additional electro-optic or acousto-optic modulator as a phase shifter for shifting the zeros of the light intensity distribution.

  As already explained, the light that influences the emission of luminescence light by means of the luminescence marker can in particular be luminescence-suppressing light that suppresses the emission of luminescence light by means of the luminescence marker outside the zero point. For example, luminescence suppression light shifts or switches a luminescent marker in the form of a switchable protein to a dark state where the luminescent marker is not excitable due to the emission of luminescent light. Luminescence suppression light excites a luminescent marker for emission of luminescence light, and in combination with excitation light having an intensity distribution with an intensity maximum in the area of the zero point of the luminescence suppression light on the sample. In particular it can be guided. This corresponds to the usual procedure in STED, RESOLFT or GSD fluorescence optical microscopy, except for a tight limit to the scan area or with a scan with increasing distance to the center point of the scan area.

  In one embodiment of the method according to the invention, the concept known from WO 2014/108455, i.e. high in the area of the intensity maximum of the induced light by switching the luminescence marker to the inactive state. Performing STED fluorescence microscopy with a switchable luminescence marker to protect the luminescence marker against intensity is applied in a modified form. In particular, before switching the scan area at the zero point, the additional switching-off light causes additional switching with an intensity distribution that switches the switchable luminescence marker to an inactive state in a partial area of the sample adjacent to the scan area. Off light is directed onto the sample. This adjacent area is close to the scanning area in at least one direction where the maximum intensity value is close to the zero point of the guide light in the sample. In this way, the luminescence marker is then switched to the inactive state, where there is a maximum intensity of the guided light, and here without this protective measure, the luminescence marker is in scanning the scanning area. Will be bleached by high intensity of induced light. In this embodiment of the method according to the invention, it can be carried out continuously for scanning areas that are in direct proximity or even overlap, in that bleaching of the luminescent markers is suppressed outside the scanning area. In other words, the sample may be scanned in a scan area, where the scan area is scanned at zeros at all positions or at least selected positions in the sample.

  The intensity distribution of the switch-off light in each adjacent area to be subsequently scanned can comprise a local intensity minimum in the scanning area formed by the destructive interference, in which it is the luminescence It does not switch the marker at least essentially off, ie it leaves the luminescent markers in their active state, at least essentially where the luminescent markers are excitable by the excitation light. Depending on the selection of the switchable luminescence marker, this active state may be switched on on each partial area of the sample to be scanned before or temporarily overlapping the light to the sample. Led, so that switchable luminescence markers need to be switched on to their active state or at least it may be preferred.

  When switched on and / or off, switchable luminescent markers often emit luminescent light. This luminescence light can be registered and evaluated. The purpose of this evaluation is, for example, whether the scanning area defined by each adjacent area is scanned at any zero point, or the luminescent light that is then emitted is registered confocally. In the meantime, the scanning area is registered while determining whether the scanning area as a whole only receives excitation light or switching on and / or off indicates that there is no relevant concentration of the luminescent marker. As a low intensity of the emitted luminescence light, it can be a determination of whether to receive any excitation light. In addition, the evaluation guides the guided light onto the sample at each position of the zero point in the scanning area defined by the adjacent partial area and registration of the luminescent light emitted from the zero point area under any conditions Can have the purpose of determining whether can be stopped properly. For example, an upper limit and / or a lower limit threshold for performing the RESCue method in each scanning area can be set according to the evaluation result.

  In one embodiment of the method according to the invention, the luminescent light emitted from the zero point area is registered with a point detector whose position relative to the sample does not change during the scanning area. This means that the movement of the zero point of the light intensity distribution of the light that affects the emission of the luminescent light by the luminescent marker is not taken into account when registering the luminescent light with the point detector. This is possible because the dimensions of the scan area are generally clearly smaller than the detection area of the point detector for the sample. This is especially true even for point detectors placed confocally with respect to the center point of the scanning area. Since the size of the scanning area is generally smaller than the diffraction barrier at the wavelength of the luminescent light, the luminescent light from the entire scanning area will reach such a point detector. A spatially fixed point sensor means that the zero point for scanning the scanning area is moved only by deflecting light that affects the emission of fluorescent light by the fluorescent marker. It is not necessary for any excitation light to be shifted by the luminescence marker with light that affects the emission of the luminescent light, since its intensity maximum typically covers the entire scanning area.

  Also as already explained, the light that influences the emission of luminescent light by means of the luminescent marker can alternatively be luminescent light that allows the emission of luminescent light by means of the luminescent marker outside the zero point. . This includes the option that this light is luminescence excitation light and is the only light that is directed onto the sample. This also includes the option that the light is luminescence activated light that moves the luminescence marker from the dark state to the excitable state for luminescence, ie activates the luminescence marker. Light that affects the emission of luminescent light by a luminescent marker can also have both functions, activation and excitation, and can have two components of different wavelengths for this purpose. If a partial area of the sample is then scanned at the zero point of the light intensity distribution that affects the emission of luminescent light by the luminescent marker, the luminescent marker in the partial area is moved to the intensity maximum value close to the zero point. This scanning area is kept small in order not to apply high light intensity in the area at all or at least as little as possible. In this embodiment of the method, the registration of the luminescent light emitted by the luminescent markers in the sample can be performed with a camera, and the evaluation is typically based on the actual position of the zero in the sample and the sample Analyzing the registered intensity distribution with respect to associated variations in the intensity distribution of the luminescent light emitted from and registered with the camera.

  The disadvantage of the method according to the invention that each scanned and therefore imaged partial area of the sample remains very small is at least partially compensated in that the sample is scanned simultaneously in several partial areas. obtain. Here, in particular, a grid of light zeros that influence the emission of luminescent light by means of luminescent markers can be used. Even so, the grid of zeros is not shifted so that the entire sample is imaged, i.e. imaged over the entire distance of the zeros in the grid. Instead, the individual partial areas in which the sample is scanned remain separated from each other even in this embodiment of the method according to the invention. Only in this case, the reduced risk of bleaching the luminescent markers in the scanned partial area is the actual scanning of the luminescent markers in the sample to protect the luminescent markers against high light intensity bleaching effects. This is achieved without further measures such as switching off the parts that are not done. In the case of the switchable luminescence markers used in this embodiment of the invention, the switchable luminescence markers are only in their fluorescent state in the respective scanning area, and the sample is further scanned in the scanning area, It will therefore be apparent to those skilled in the art that it can be fully imaged.

  If several copies of the object of interest are each placed overlapping one or more scanning areas, a partial image of this object is obtained by scanning each scanning area. If these partial images are statistically distributed across the object, and if their number is high enough, a complete image of the entire object of interest can be reconstructed from the partial images. It is clear that this reconstruction requires that several copies of the object of interest need to be at least essentially equivalent. In order to assign the partial images to specific points of the object of interest, copies of the object of interest in the sample can be further imaged in another way to determine their position and orientation relative to the scan area. The object of interest placed in several copies in the sample can be, for example, a molecule or a virus. Furthermore, in this embodiment of the method according to the invention, multiple copies of the object of interest may be subject to varying ambient conditions and register the object's response to these varying conditions. For this purpose, the individual scan areas are scanned at their respective zeros during and / or before and after changes in ambient conditions. The sequence of continuous scanning of the scanning area at each zero can be quite high so that even fast changes of the object of interest can be registered.

  A scanning luminescence optical microscope according to the present invention provides light on a sample with an intensity distribution having a light source for light that affects the emission of luminescent light by a luminescence marker, and an intensity maximum near the zero. A light shaper for directing to a light source, a scanner for scanning a partial area of the sample to be scanned at the zero point, a detector for registering luminescent light emitted from the zero point area, and one of the methods according to the invention And a controller for performing the operation.

  The detector can be a point detector and the position of the point detector can be fixed relative to the sample while scanning the scan area. This means that since the scanning area generally has dimensions well below the diffraction barrier, the detector can detect the luminescent light emitted from the sample without descanning the luminescent light. In that case, unlike a scanner for scanning the scanning area at the zero point, a further scanner configured to scan a larger area of the sample in at least one direction may be provided.

  A scanner for scanning a larger area of a sample in at least one direction may include a sample holder or stage that is movable relative to the objective lens of the light shaper, but the sample to be scanned in at least one direction The scanner for scanning the partial area may include an electro-optic scanner, an acousto-optic deflector, a galvo scanner, or a galvo mirror.

  The detector is, for example, a point detector that registers descanned luminescence light emitted from the sample, or a two-dimensional camera such as a camera that registers luminescence light that is not descanned at a fixed relative position with respect to the sample. It can be a detector.

  In the scanning luminescence optical microscope according to the invention for performing the STED method according to the invention, the light provided by the light source is a guide light, there is a further light source providing excitation light, and the light shaper is a luminescence suppressor. The excitation light is guided onto the sample with an intensity distribution having a maximum value in the area of the light zero point.

  In order to perform the method according to the invention utilizing a switchable luminescence marker, an additional switch-off light source for switch-off light should be provided in the scanning luminescence optical microscope, the light shaper being scanned Switch-off light is directed onto the sample in an intensity distribution such that it switches off the switchable luminescence marker in an inactive state in the partial area of the sample proximate to the partial area to be. This adjacent area is close to the scanning area in at least one direction where the maximum intensity value is close to the zero point of the guide light in the sample. Furthermore, a switch-on light source for switch-on light that switches the switchable luminescence markers to their active state can be provided, the light shaper scanning before or temporarily overlapping with the switch-off light. Switch-on light is guided onto the sample in the partial area including the area.

  In experiments to test the method according to the invention, a> 100-fold increase in the yield of photons from luminescent markers in the sample was achieved. This means that to record changes in the structure of interest in the sample, 100 times more images can be taken from that changing structure. Further, the more photons are emitted continuously per time unit by the luminescent marker in the sample, the less time is required for each individual image.

  Turning now to the drawings in more detail, FIG. 1 shows a section through the intensity distribution of the excitation light 1 at the top thereof. The excitation light 1 has an intensity maximum value 27 of the maximum intensity I at the geometric focus F. However, the intensity I is distributed over an area having dimensions that extend far beyond the focal point F in all directions. The diameter of this area corresponds to the diffraction barrier at the wavelength λ of the excitation light 1 and the numerical aperture NA of the objective lens used for focusing the excitation light 1 at the focal point F according to λ / NA. In order to limit the effective excitation of the luminescent marker in the sample to an area where the intensity distribution of the excitation light 1 is smaller than the area over which it extends, a fluorescence suppression having a zero 4 and an intensity maximum 3 close to the zero 4 Light 2 is further directed onto the sample. The luminescence suppression light 2 suppresses the emission of the luminescence light by the fluorescence marker excited by the excitation light 1 in that the fluorescence marker is back-excited again by stimulated emission, for example. Since the intensity I of the fluorescence suppression light 2 is very high everywhere outside the area 5 of the zero point 4 of the fluorescence suppression light 2, this reverse excitation is complete, i.e. the luminescent marker located there does not emit any fluorescence light. . On the contrary, the term “zero point 4” refers to the entire area 5, and in this area 5, the intensity I of the fluorescence suppression light 2 remains very small, so that it is reflected by the fluorescent marker located here. Release is not at least completely suppressed. In the lower part, FIG. 1 shows the spatial distribution of the effective fluorescence excitation 6. This effective fluorescence excitation 6 is limited to the area 5 of the zero point 4. When the sample is scanned at zero point 4, the fluorescent light emitted from the sample always comes from area 5 and can therefore be assigned to the location of area 5 in the sample.

  When the zero point 4 while scanning the sample is closer to the structure of interest marked with a fluorescent dye, i.e. a fluorescent marker, the fluorescent marker is first intensified before the fluorescent marker enters the area 5 of the zero point 4. It enters the area of the maximum value 3 and the intensity on which the excitation light 1 is superimposed. In particular, while scanning the sample line by line, the fluorescent marker repeatedly receives high light intensity before the fluorescent marker enters area 5 and the fluorescent light emitted by the fluorescent marker is registered for the first time. This can mean that the fluorescent marker has already been bleached before it enters the area 5 for the first time. Due to this effect, it is often impossible to scan the same sample repeatedly, for example to monitor temporal changes in structures of interest marked with fluorescent markers in the sample.

However, as shown in FIG. 2, if the scanning area of the sample scanned at zero 4 is defined as no more than 3/4 or 75% of the distance D _{0 with} an intensity maximum of 3, the excitation according to FIG. In combination with the intensity of light 1, the average stress of the fluorescent marker in the scanning area caused by the high intensity of the fluorescence suppression light 2 in the area of maximum intensity is already reduced. This stress is further reduced if the size of the scanning area is limited to less than half of the distance D _{0 with} a maximum intensity value of 3. To limit the size to less than D _0/2, when scanning the scan area, the point of the scan area does not enter the peak area of maximum intensity 3. If the dimensions of the scan area is limited to D _0/4, the maximum intensity of the fluorescence suppression beam 2 sample is subjected in the scanning area is reduced to about I _0/2, however, I ₀ is the intensity maximum 3 This is the maximum intensity of the fluorescence suppression light 2.

FIG. 3 shows a scanning area 7 of the scanning sample 3, shown here only partly here in the area 5 of the zero point 4 along the course 8, here in the form of a meander. The partial area 7 to be scanned is shown within the intensity maximum value 3 and the position of these intensity maximum values 3 corresponds to the alignment of the zero point 4 with the center point 10 of the scanning area 7. Correspondingly, the intensity maximum, or more precisely, is highlighted in FIG. 3 and extends around the position of the zero point 4 indicated by the dashed line 11, where the ring-shaped intensity maximum 3 is It still overlaps with the scanning area 7. However, this overlap, the size of the scan area 7 can be avoided by further limited to less than D _0/2. However, even by limiting the size of the partial area 7, as shown here about 2D _0/3, a significant reduction in the average load of the fluorescent marker in the scan area 7 by fluorescence depletion light 2 is achieved The

FIG. 4 shows the scan area 7 of the sample 8 reduced to D _0/2 . Here, the ring-shaped course of the maximum intensity value 3 indicated by the broken line 11 no longer reaches the partial area 7 at any position of the zero point 4 in the scanning area 7. Furthermore, FIG. 4 shows a spiral course 12 in which the scanning area 7 starts at the center point 10 and is scanned along it. When scanning the scanning area 7, the fluorescent light emitted and registered from the sample 8 is assigned to the respective location of the zero point 4 in the sample 3, regardless of the course shape along which the zero point 4 is moved. .

  The embodiment of the invention shown in FIG. 5 is shown in FIGS. 3 and 4 in that the scanning area 7 is no longer limited to a piece of what is surrounded by a ring-shaped maximum 3 around the center point 10. Different from the described embodiment. Instead, a partial area 8 of the sample is scanned along the spiral course 12 and its diameter is larger than the diameter of the ring-shaped maximum 3 around the center point 10. Here, the previous exposure to the fluorescence suppression light 2 of the part of the scanning area 7 which is only traversed later by the spiral course 12 steadily increases. In the part close to the center point 10, the previous load due to the fluorescence suppression light 2 is, however, minimal. If the center point 2 of the scanning area 7 is led to a particularly interesting point of the sample 8, the maximum yield of fluorescent light from the sample 8 is now achieved and this maximum yield decreases with increasing distance to the center point 10. However, it remains sufficient to image around a particularly interesting point of sample 8.

  FIG. 6 shows a scanning fluorescence optical microscope 13 which is particularly suitable for carrying out the method according to the invention. The scanning fluorescence optical microscope 13 includes a light source 14 that provides fluorescence suppression light 2, and the cross section of the fluorescence suppression light 2 is widened by the magnifying optical component 15, and the wavefront of the fluorescence suppression light 2 that crosses the cross section of the light is suppressed. The phase plate in such a way that when the light 2 is focused on the sample 8 by the objective lens 45, the zero point 4 and the adjacent intensity maximum 3 according to FIGS. 1 and 2 are formed around the respective focal point F. 16 is modulated. A further light source 17 with a further magnifying optic 18 provides the excitation light 1. By means of the dichroic mirror 19, the excitation light 1 and the fluorescence suppression light 2 are combined so that the excitation light 1 has its maximum intensity value 27 in the area 5 of the zero point 4 of the fluorescence suppression light 2 according to FIG. The fluorescent light 20 emitted from the zero point area of the fluorescence suppression light 2 is separated by the dichroic mirror 26, registered by the point detector 21, and assigned to each location of the zero point 4 in the sample 8. Scanners 22 and 23 are provided for two orthogonal scan directions, and scanners 22 and 23 are operated in combination to scan the respective scan area in sample 8 at the zero point of fluorescence suppression light 2. The scanners 22 and 23 only affect the direction of the excitation light 1 and the fluorescence suppression light 2; the scanners 22 and 23 can even be arranged only in the beam path of the fluorescence suppression light 2. When the scanning area 7 of the sample 8 has a dimension below the diffraction barrier, the fluorescent light 20 emitted from the zero point area of the fluorescence suppression light 2 in the scanning area in the sample 8 is a point detector 21 for the sample 3 Is always in the point detector 21 even in the case of a spatially fixed arrangement, ie despite the zero shift by the scanners 22 and 23. This is because the scanning area 7 has a dimension below the diffraction barrier. For example, in order to initially determine the position of the preferred scan area, to scan the sample 8 beyond the scan area 7, a further scanner, simply indicated here by the corresponding shift symbol 25, is Provided throughout the area.

  Up to this point, it has not yet been explicitly stated that the zero point 4 of the intensity distribution of the fluorescence suppression light 2 can also be defined by the maximum intensity value 3 that is close in the z direction, where the fluorescence suppression light 2 is In order to increase the spatial resolution when imaging the structure of interest in the sample 8 also in the z direction, it is guided onto the sample. Correspondingly, the scanning area 7 will then also be limited in this z direction to no more than 75%, preferably less than 50% of the distance of the light intensity maximum in the z direction, or Starting at the center point 10 and with increasing distance to the center point 10 will be scanned in the z direction. The increase in spatial resolution when imaging a sample in the z-direction is known in the field of fluorescence microscopy, for example, other means such as 4PI placement of fluorescent markers or two-photon excitation to emit fluorescent light. It can also be achieved by other means. It is also generally true that the methods described herein can be supplemented by other means known in the field of fluorescence microscopy. Applying fluorescence suppression light 2 and / or excitation light 1 in pulses, simultaneous and continuous application of excitation light 1 or fluorescence suppression light 2, and gate registration of fluorescence light at defined time gates after each pulse, etc. Belonging to the means.

  For the sample, a confocal image according to FIG. 7 (a) has been taken and the structure of interest is marked with the luminescence marker nucleoporin gp210. Confocal images provide an overview of the structure of interest. From this overview, separate scan areas have been selected in which STED images have been taken according to the present method of the present invention. These scanning areas are smaller than the focal area of the excitation light. In the scanning area, the structure of interest is imaged with both high spatial resolution and high yield of fluorescent light. To image a partial image of the sample shown in FIG. 7 (b) and present a scanning area, excitation light at a wavelength of 635 nm and a power of 5 μW is pulsed into the sample at a repetition rate of 20 MHz. . STED light at a wavelength of 775 nm is guided to the sample with a sync pulse at a pulse length of 1.2 ns at a power of 150 mW. The excitation light and STED light are focused on the sample by the 1.4NA oil reversal objective lens. The fluorescent light is focused on the point detector by an oil reversal objective lens and a further lens.

FIG. 8 shows the bleaching behavior of the stained nuclear pore protein complex as a function of the size of the scanning area in STED scanning fluorescence optical microscopy. τ1 / 2 indicates the number of images that can be taken before the fluorescent signal drops to half the starting value due to bleaching. τ1 / 2 is plotted in nanometers over the dimensions of the scan area. The STED power was 160 mW and the excitation power was 2 μW. Otherwise, the STED conditions corresponded to the conditions according to FIG. With a scan area size of 100 × 100 nm ² , bleaching is reduced by a factor of 100 compared to a size of 800 × 800 nm ² . Correspondingly, for example, 100 times more images can be taken from the same scanning area to monitor the dynamic process in the sample.

  The scanning fluorescent microscope 13 according to FIG. 9 has the following differences from the scanning fluorescent microscope shown in FIG. When viewed from the sample 8, the point detector 21 is placed behind the scanners 22 and 23 so that the scanner descans the fluorescent light 20 coming from the sample 3 toward the detector 21. Here, the scanners 22 and 23 are provided both for scanning the scanning area to be scanned at the zero point of the fluorescence suppression light 2 and for arranging and shifting the scanning area entirely in the sample 8. The FIG. 9 also shows a detector 28 for fluorescent light located upstream of the scanners 22 and 23 when viewed from the sample 8. However, this is not a point detector, but a camera 29, ie a two-dimensional detector. This detector 28 may be provided in addition to or in place of the point detector 21, and a dichroic mirror 30 that deflects the fluorescent light 20 towards the detector 28 includes an objective lens 45 and a scanner 22. And 23 temporarily or permanently.

  Further, the scanning fluorescence microscope 13 according to FIG. 9 has a switch-off light source 31, which provides a switch-off light 34 before scanning each scanning area at the zero point of the fluorescence suppression light 2. The magnifying optical component 32 is assigned to do this. The switch-off light 34 is coupled in by a dichroic mirror 43 and its intensity distribution in the sample 8 is in the sample 8 in a partial area of the sample 8 where the beam shaper 33 is close to the partial area to be scanned. Is formed by the beam shaper 33 to switch the switchable luminescence marker at In this inactive state, the switchable luminescence marker in sample 8 is not excitable due to the emission of fluorescent light 20 by excitation light 1. Correspondingly, the fluorescence-inhibiting light 2 in the form of guided light for the luminescent markers in the area close to the scanning area eliminates the risks associated with bleaching. 9, the scanning fluorescence microscope 13 according to FIG. 9 scans a further scanning area of the sample 8 close to the already scanned scanning area of the sample 8 at the zero point of the fluorescence suppression light 2, and the fluorescence emitted from the sample 3. It is possible to register light 20 because luminescent markers in further scanning areas were inactive, protecting them against bleaching, so This is because it has not been bleached by scanning before.

  In order to be able to excite the luminescence marker in the scanning area adjacent to the excitation light 1 for the emission of the fluorescent light 20, the luminescence marker needs to be in an active state. To accomplish this, the luminescence marker may wait for spontaneous return from the inactive state to the active state. However, the scanning fluorescence microscope 13 according to FIG. 9 provides an additional switch-on light source with magnifying optics 36 that provides the switch-on light 37 and directs the switch-on light 37 onto the sample 8 via the dichroic mirror 44. 35 is also provided. With the switch-on light, the luminescent markers in the next scanning area are initially switched to their active state. The partial area of sample 8 covered by the switch-on light 37 is then transferred to their inactive state by the switch-off light 34 since the luminescence markers outside the next scan area to be scanned are moved to their inactive state. Can be larger than the scanning area to be scanned. Thus, the sample 8 is scanned using the scanning fluorescence microscope 13 in two steps: a large step using the scanning area and a small step using the zero point of the fluorescence suppression light 2 within each location of the scanning area. obtain.

  When switching the luminescence marker in the sample 8 on and / or off with the switch-on light 37 or the switch-off light 34, various switchable luminescence markers are also excited for the emission of the fluorescent light 20. This fluorescent light 20 thus already provides information on the concentration of the luminescent marker in each partial area of the sample 8. This information can correspondingly be evaluated and used to determine whether there is any value in scanning the next scanning area at the zero point of the fluorescence suppression light 2. In the absence of any value, such a scan will not be performed so that the sample 8 is not unnecessarily exposed to the fluorescence suppression light 2. Furthermore, the fluorescence light registered while switching the luminescence marker on and off limits the exposure of the sample 8 to the fluorescence suppression light 2 and the excitation light 1 as soon as possible according to the RESCue method. In order to do this, it can be used to set an upper and / or lower threshold for the fluorescent light registered at each location of the zero point of the fluorescence suppression light 2 in the scanning area.

  Importantly forgotten, FIG. 9 shows a controller 38 for the light sources 14 and 17, the switch-on light source 35, the switch-off light source 31 and the scanners 22 and 23 of the scanning fluorescence microscope 13. The controller 38 controls them to carry out the method according to the invention.

  FIG. 10 shows the scanning area 7 and the maximum fluorescence suppression light intensity value 3 surrounding the partial area 7, and the zero point of the fluorescence suppression light is at the center point 10 of the scanning area 7. Further, FIG. 10 shows a ring-shaped adjacent partial area 39 in which the sample 8 receives the switch-off light 34 before scanning the scanning area 7, thereby The switchable fluorescent markers located at are moved to their inactive state. The partial area 39 excludes the scanning area 7, i.e. the intensity of the light-off switch 34 is zero or at least very small in the scanning area 7, so that within the time period during which the switch-off light 34 is guided to the sample 8. Insufficient to switch off the luminescent marker. In order to ensure that the switchable luminescence markers are in their active state, at least in the scanning area 7, the switch-on light 37 is scanned before or temporarily overlapping the switch-off light 34. It is guided on the sample 8 in a circular partial area 40 including the area 7.

  FIG. 11 shows how the sample 8 can be scanned in the scanning area 7. Here, FIG. 11 (a) shows several successive positions of the circular scanning area 7 according to FIG. 10 in the sample 8; for one of these positions of the scanning area 7, the square area of the sample 8 is A course 9 scanned along it is shown in the partial area 7. FIG. 11 (b) shows how the sample 8 is completely covered with such a square area 41 and can therefore be fully imaged.

  FIG. 12 shows another method of scanning the sample 8 in the scanning area 7. Here, the continuous positions of the circular scanning area 7 are arranged in a hexagonal arrangement in the sample 8. The course 9 in which the partial area 7 is scanned along the zero point of the luminescence suppression light at each of its positions extends over a regular hexagon. FIG. 12 (b) shows how these regular hexagons 42 can be used to cover the entire sample 8 and thus be imaged.

  FIG. 13 is a block diagram of a method according to the present invention, beginning with step 46 which determines the scan area 7 with respect to both the size of the scan area 7 in the sample 8 and the location of the scan area 7. In the next step 47, the scanning area 7 is scanned at the zero point 4 of the intensity distribution of the fluorescence suppression light 2. In parallel step 48, the fluorescent light emitted from the sample is registered. In step 49, the fluorescent light registered in step 48 is assigned to the current location of zero 4 in step 47. This implies that in step 48 the fluorescent light emitted from the sample 8 is registered with temporal resolution and in step 47 that the scanning area 7 is scanned with zero point 4 in temporal resolution, thus The specific amount of fluorescent light registered in step 48 can be assigned to a specific position of the zero point 4 in the scanning area 7. Through steps 47 to 49, the scanning area 7 is scanned and imaged once. This imaging can be repeated, for example, to monitor changes in the structure of interest marked with a fluorescent marker in the scanning area 7.

  The method embodiment of the present invention shown in FIG. 14 begins at step 46 where the scan area 7 is determined. In a subsequent step 50, the fluorescent marker in the area including the scanning area 7 is switched on. This implies, for example, that the fluorescent marker is a switchable marker that can be switched on by the switch-on light 37. In the next step 51, the fluorescent marker outside the scanning area 7 is switched off to the inactive state. This can be done by the switch-off light 34. In the inactive state, the fluorescent marker is protected against bleaching due to the high light intensity of the fluorescence suppression light 2. Next, in step 47, the scanning area 7 is scanned at the zero point 4 of the intensity distribution of the fluorescence suppression light. Thereafter, step 46 is repeated. In this repetition of step 46, the scanning area 7 may be placed at a position close to the previous position of the scanning area 7. The fluorescent marker now receives a high intensity of fluorescence suppression light, but since the fluorescent marker was in an inactive state, the fluorescent marker is not bleached here. Thus, the fluorescent marker can now be measured. The loop including steps 46, 50, 51 and 47 is then repeated until all areas of interest in the sample are scanned in scan area 7 and thus imaged.

  Many changes and modifications may be made to the preferred embodiments of the present invention without substantially departing from the spirit and principles of the present invention. All such modifications and changes are intended to be included herein within the scope of the present invention as defined by the following claims.

Claims (27)

A method for high resolution imaging of a structure in a sample, wherein the structure is marked with a luminescence marker, the method comprising:
Directing light on the sample that influences the emission of luminescent light by the luminescence marker in an intensity distribution having a zero and an intensity maximum close to the zero in at least one direction;
Scanning a scanning area at the zero point, the scanning area being part of the sample;
Registering luminescent light emitted from a local area including the zero in the sample while scanning the scanning area;
Assigning the registered luminescent light to each location of the zero in the sample;
Including
The dimension of the scanning area in the at least one direction where the intensity maximum is close to the zero in the sample is not greater than 75% of the distance of the intensity maximum in the at least one direction;
Method.   The dimension of the scanning area in the at least one direction where the intensity maximum is close to the zero in the sample is not greater than 25% of the distance of the intensity maximum in the at least one direction. Item 2. The method according to Item 1.   The dimension of the scanning area in the at least one direction where the intensity maximum is close to the zero in the sample is such that the intensity of the light in the at least one direction starts at the zero, over which The method of claim 1, wherein the distance is not greater than a distance increasing to 25% of the intensity of the light at the adjacent intensity maximum.   The method of claim 1, wherein the scan area is repeatedly scanned at the zero point.   The method of claim 1, wherein prior to scanning the scan area, the structure in the sample is an image in another method for determining the position of the scan area in the sample. Prior to scanning the scan area, a larger partial area of the sample is
An intensity at least 50% lower than the light;
6. The method of claim 5, wherein the zero is scanned at at least one of at least 50% higher scan speed.   6. The method of claim 5, wherein a scanner is used to scan the scan area, and another scanner is used to scan the larger area of the sample. A sample holder is moved relative to an objective lens by which the light is directed onto the sample to scan the larger area of the sample;
Electro-optic scanner,
Acousto-optic deflector,
At least one of a galvo scanner and a galvo mirror is used to scan the scanning area in the at least one direction;
The method of claim 5.   The method of claim 1, wherein the light is luminescence suppression light whose wavelength is selected to suppress the emission of luminescence light by the luminescence marker outside the zero point.   The luminescence suppression light is guided light whose wavelength is selected to suppress the emission of the luminescence light by the luminescence marker outside the zero point by stimulated emission, Excitation light whose wavelength is selected to excite the luminescence marker for emission, with excitation light having an intensity distribution including a maximum value in the area of the zero point of the luminescence suppression light, The method of claim 9, wherein the method is directed onto a sample.   Prior to scanning the scanning area at the zero point, additional switch-off light is applied to the sample in an intensity distribution such that the switch-off light switches the luminescence marker to an inactive state in the adjacent area. 11. The method of claim 10, wherein the adjacent area is adjacent to the scanning area in the at least one direction where the maximum intensity is close to the zero point of the guided light in the sample.   The method of claim 11, wherein the sample is scanned in the scan area, and the scan area at all or selected locations of the scan area in the sample is scanned at the zero point.   The method of claim 11, wherein the intensity distribution of the switch-off light includes a local intensity minimum formed by destructive interference in the scan area.   Prior to or temporarily overlapping the switch-off light to the sample, the switch-on light is directed onto the scanning area of the sample that switches the luminescence markers to their active state. Item 12. The method according to Item 11.   15. A method according to claim 14, wherein the luminescence light emitted by the switchable luminescence marker is registered and evaluated when switched on or off. The result of the step to evaluate is:
Whether the scanning area defined by the adjacent area will be scanned at the zero point;
Whether the excitation light will be directed onto the sample in the scanning area defined by the adjacent partial area; and under what conditions the guided light is defined by the adjacent area At least one of guiding on the sample at each position of the zero in the scanning area and registering the luminescent light emitted from the area of the zero will be interrupted. 16. The method of claim 15, wherein   The method of claim 1, wherein the luminescent light emitted from the area at the zero point is registered with a point sensor that remains unchanged relative to the sample while scanning the scanning area.   The method of claim 1, wherein several scan areas of the sample are scanned simultaneously.   Several copies of the object of interest are placed in the sample with overlap with the several scan areas, and the image of the object of interest is acquired when scanning the several scan areas The method of claim 18, comprising a partial image of an object. A method of high spatial resolution imaging of a structure in a sample, wherein the structure is marked with a luminescence marker, the method comprising:
Directing light on the sample that influences the emission of luminescent light by the luminescence marker in an intensity distribution having a zero and an intensity maximum close to the zero in at least one direction;
Scanning a scanning area at the zero point, the scanning area being part of the sample;
Registering luminescent light emitted from a local area including the zero in the sample while scanning the scanning area;
Assigning the registered luminescent light to each location of the zero in the sample;
Including
The scan area is scanned at the zero point starting at a center point and with increasing distance to the center point in the at least one direction;
Method.   21. The method of claim 20, wherein the scan area is scanned along a spiral course around the center point. A scanning luminescence optical microscope,
A light source configured to provide light;
A light shaper configured to direct the light onto the sample in an intensity distribution having a zero and an intensity maximum proximate to the zero;
A scanner configured to scan a scanning area at the zero point, wherein the scanning area is a partial area of the sample;
A detector configured to register luminescent light emitted from the area of the zero;
A controller programmed with software implementing a method for high resolution imaging of structures in a sample, wherein the structure is marked with a luminescence marker, the method comprising:
Directing light on the sample that influences the emission of luminescent light by the luminescence marker in an intensity distribution having a zero and an intensity maximum close to the zero in at least one direction;
Scanning a scanning area at the zero point, the scanning area being part of the sample;
Registering luminescent light emitted from a local area including the zero in the sample while scanning the scanning area;
Assigning the registered luminescent light to each location of the zero in the sample;
Including
The dimension of the scanning area in the at least one direction where the intensity maximum is close to the zero in the sample is not greater than 75% of the distance of the intensity maximum in the at least one direction, or A scanning area is scanned at the zero point starting at the center point and with increasing distance to the center point in the at least one direction,
Scanning luminescence optical microscope.   Further comprising a further scanner configured to scan a larger area of the sample, wherein the detector is a point detector whose position relative to the sample is fixed while scanning the scanning area. Item 23. The scanning luminescence optical microscope according to Item 22.   24. A scanning luminescence optical microscope according to claim 23, wherein the further scanner comprises a sample holder movable relative to the objective lens of the light shaper.   The scanning luminescence optical microscope according to claim 22, wherein the scanner comprises at least one of an electro-optic scanner, an acousto-optic deflector, a galvo scanner, or a galvo mirror. The light provided by the light source is guided light and the scanning luminescence optical microscope is:
A further light source configured to provide excitation light, wherein the light shaper directs the guided light onto the sample with an intensity distribution having a maximum value in the area of the zero point of the guided light. A configured light source;
A switch-off light source configured to provide switch-off light, wherein the light shaper causes the light shaper to switch off a switchable luminescence marker in an adjacent partial area to an inactive state. The switch-off light is configured to be guided onto the sample with a strong intensity distribution, and the adjacent partial area is located in the scanning area in the at least one direction where the maximum intensity value is close to the zero point of the guide light. The scanning luminescence optical microscope according to claim 23, further comprising an adjacent switch-off light source.   Further comprising a switch-on light source configured to provide switch-on light to switch the switchable luminescence markers to their active state, the light shaper prior to directing the switch-off light onto the sample. 27. The scanning luminescence optical microscope according to claim 26, wherein the scanning luminescence optical microscope is configured to guide the switch-on light onto the scanning area.